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Method for the removal/purification of serum albumins and means for use in the method

a serum albumin and purification technology, applied in the field of method for removal/purification of serum albumin and methods for use, can solve the problems of less suitable as ligands

Inactive Publication Date: 2003-09-02
GE HEALTHCARE BIO SCI CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The first objective of the invention is to provide improved affinity methods for the removal of a serum albumin from a mixture of proteins in order to produce the serum albumin in pure form or a preparation essentially free of the removed serum albumin.
A third objective is to provide new affinity matrices carrying albumin-binding ligands having improved selectivities for serum albumins.

Problems solved by technology

The various extra binding abilities of these proteins make them less suitable as ligands for the selective / specific removal of serum albumin from complex protein mixtures.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

Immobilisation: The protein used in this study was a 7.1 kDa fragment of Streptococcal protein G containing the complete albumin binding domain, A3 (fragment SPG-ABD3, aa 254-299 in Kraulis et al., FEBS.letter 378 (1996) 190-194). Approximately 2 mg of the fragment was immobilised on a 1 ml NHS-activated column, HiTrap, according to the manufacturers instruction (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (NHS=N-hydroxy-succinimide).

Chromatography conditions: All samples were adjusted to pH 7.0 before loading onto the column. A buffer consisting of 20 mM sodium phosphate and 150 mM sodium chloride, pH 7.0, was used for equilibration and washing after sample loading on the column (Buffer A).

Elution was performed by decreasing pH to 2.7 by applying a 20 mM citrate, 150 mM sodium chloride buffer, pH 2.7 (Buffer B).

Samples: Human serum albumin HSA (Sigma, St Louis, Mo, U.S.A.) 2 mg / ml in Buffer A, bovine serum albumin BSA (Sigma, St Louis, Mo, U.S.A.) 2 mg / ml in Buffer A and acid t...

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Abstract

A method for removing a serum albumin from a mixture of other compounds by contacting said mixture with a ligand a) having affinity for and enabling selective binding of the serum albumin and b) being attached to a base matrix insoluble in the aqueous media used or being possible to attach to such a matrix after having become bound to the serum albumin, characterized in that said ligand is derived from an albumin binding bacterial cell surface receptor and that the ligand lacks the IgG-binding and / or alpha2-macroglobulin-binding ability found in native forms of these type of bacterial receptors. An albumin-binding ligand derived from a cell surface bacterial receptor and attached covalently to a carrier matrix, characterized in that the ligand is monovalent with respect to ability to bind a serum albumin. A method for removal of serum albumin from a sample that is to be assayed for non-serum albumin components. The characteristic feature is to subject the sample to affinity adsorption by an albumin-binding ligand derived from an albumin-binding receptor.

Description

1. Field of the InventionThe present invention concerns a method for the separation / removal of a mammalian serum albumin from a solution containing a mixture of proteins in order to obtain a solution / preparation that is substantially devoid of the serum albumin. The invention also concerns novel immobilized forms of albumin-binding ligands deriving from native forms of bacterial receptors that are able to bind to one or more serum albumins.2. Technical BackgroundFor a long time there has been a large demand for mammalian serum albumins, for instance serum albumin of human or bovine origin (HSA and BSA, respectively). For HSA this has mainly depended on its therapeutic use as a plasma volume expander. Originally serum albumins were obtained from sera / plasma of the appropriate species origin. For some years the focus has been to produce serum albumins recombinantly, in particular HSA. For bacterially produced recombinant forms, it has become urgent to remove host cell contaminants bec...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K14/435C07K14/765C12N15/09C07K14/705
CPCC07K14/765
Inventor JOHANSSON, HANS
Owner GE HEALTHCARE BIO SCI CORP
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